Skin- and airway-deliverable TRPA1 inhibitor.

This study explored the potential of perfumery compounds as sources of transient receptor potential ankyrin 1 (TRPA1) inhibitors that could be formulated for effective delivery to the skin and airways. A highly potent, small, and selective TRPA1 inhibitor, 2-methyl-4-phenyl-1-pentanol (1), was discovered in perfumery compounds. Compound 1 demonstrated promising inhibitory activity against a broad range of TRPA1 agonists. A single stereoisomer of 1 was identified as the most effective TRPA1 inhibitor, indicating the potential for stereoselective synthesis to enhance its potency. Additionally, the structure-activity relationship of 1 was evaluated to elucidate the structural features of TRPA1 inhibitors within the fragrance-like compounds. Notably, the topical application of 1 alleviated sensory irritation in individuals with sensitive skin, while the inhalation of 1 resulted in a significant reduction in ammonia irritation, underscoring its efficacy in both skin and airway applications.

Structure-activity relationship studies on an antitumor marine macrolide using aplyronine a-swinholide A hybrid.

An aplyronine A-swinholide A hybrid, consisting of the macrolactone part of aplyronine A and the side chain part of swinholide A, was designed, synthesized, and biologically evaluated. This hybrid induced protein-protein interactions between two major cytoskeletal proteins actin and tubulin in the same manner as aplyronine A, and exhibited potent cytotoxicity and actin-depolymerizing activity. The importance of the methoxy group in the N,N,O-trimethylserine ester was clarified by the structure-activity relationship studies of the amino acid moiety by using the hybrid analogs. Furthermore, the comparison of the actin-depolymerizing activities between the side chain analogs of aplyronine A and swinholide A showed that the side chain analog of swinholide A had much weaker actin-depolymerizing activity than that of aplyronine A.

Isolation and Synthesis of Azuriaplysins A and B, Bromoditerpenes with an α-Methylene Carbonyl from the Sea Hare Aplysia kurodai.

New bromoditerpenes having an α-methylene carbonyl structure, azuriaplysins A (1) and B (2), were isolated from the sea hare Aplysia kurodai. Their relative stereostructures were determined based on one- and two-dimensional NMR spectroscopic analysis. In addition, the absolute stereostructures were determined by the total synthesis of both enantiomers of azuriaplysins A (1) and B (2), the key points of which were bromocyclization of farnesol and optical resolution of a key intermediate. Azuriaplysin B (2) and its enantiomer exhibited moderate cytotoxicity against HeLa S3 cells.

Structure Revision of Trichomide D by Total Synthesis.

A structure revision of trichomide D has been achieved by its total synthesis. The sterically hindered peptide sequence was successfully prepared using not only a conventional amidation with EDCI but also coupling with an Fmoc-protected amino acid chloride derivative. The cyclization precursor was synthesized by coupling of a tetrapeptide with an acylproline derivative and subsequent removal of silyl groups at the N- and C-termini. Macrolactonization using MNBA/DMAPO followed by preparation of a chlorohydrin moiety furnished the proposed structure of trichomide D, whose spectra were not identical to those of the natural product. Finally, we succeeded in the elucidation of the true structure of trichomide D by its total synthesis, and the absolute configuration of the chlorohydrin moiety was revised to be S. The cytotoxicities of the natural product and its synthetic derivatives against MCF-7 and HeLa S3 cells were evaluated by the MTT method, revealing that the configuration of the chlorohydrin moiety is a pivotal factor for exhibiting potent cytotoxicity against cancer cells.

Specific protein-labeling and ligand-binding position analysis with amidopyrene probes as LDI MS tags.

To readily analyze the binding mode of protein-ligand interactions, we developed ligand-bound-type and ligand-dissociation-type probes having 6-amidopyrene (apy) as a detecting group. Matrix- and label-assisted laser desorption/ionization mass spectrometry (MALDI and LA-LDI MS) analyses and a covalent docking simulation using these probes precisely determined the binding position of the ligand biotin on the target protein avidin (RMSD = 0.786 and 0.332 Å). Our apy-probe-labeling method may be useful for determining the unknown ligand-binding sites of various target proteins.

Development of photoaffinity derivatives of the antitumor macrolide aplyronine A, a PPI-inducer between actin and tubulin.

The antitumor and actin-depolymerizing marine macrolide aplyronine A (ApA) synergistically binds to tubulin in association with actin, and prevents spindle formation and mitosis. While the crystal structure of the actin ApA complex was solved in 2006, its interaction with the tubulin heterodimer has not been clarified. To investigate the binding modes of ApA as a unique protein-protein interaction (PPI)-inducer between these two cytoskeletal proteins, we prepared its photoaffinity acetylene and fluorescent derivatives with the aid of molecular modeling studies for probe design. Among these three derivatives, the ApA-PPA-TAMRA probe specifically photoreacted with both actin and tubulin in vitro. However, the photolabeling yield of tubulin was quite low (up to ∼1%), and one of the major side-reactions was the addition of a water molecule to the carbene species generated from an aryldiazirine moiety on the hydrophilic surface of actin.

Binding position analysis of target proteins with the use of amidopyrene probes as LA-LDI enhancing tags.

Amidopyrene-conjugated compounds can be detected by label-assisted laser desorption/ionization mass spectrometry (LA-LDI MS) without matrixes. When actin, a cytoskeletal protein, was labeled with an excess amount of amidopyrene N-hydroxysuccinate (apy-OSu), eight apy-labeled actin peptides were predominantly detected by LA-LDI MS. Then actin was labeled with an amidopyrene NHS ester of the antitumor marine macrolide aplyronine A (ApA-apy-OSu) to form a 1 : 1 conjugate. The sequence of an apy-labeled peptide was established as A(108)PLNPKANR(116) by MS/MS analysis, in which the NHS ester moiety specifically reacted with the ε-amino group of K113. While the fragmentation at the linker part reduces the detection sensitivity of apy-labeled peptides on LA-LDI MS, our chemical probe method is useful for analyzing the binding modes of various ligands and target biomacromolecules that include multiple and weak interactions.

Total synthesis of natural derivatives and artificial analogs of 13-oxyingenol and their biological evaluation.

We have established an efficient synthetic methodology for the 13-oxyingenol natural derivative (13-oxyingenol-13-dodecanoate-20-hexanoate), featuring a ring-closing olefin metathesis reaction for the "direct" construction of a highly strained inside-outside framework and a Mislow-Evans-type [2,3]-sigmatropic rearrangement for the stereoselective introduction of the hydroxy group at C5. We also synthesized artificial analogs of 13-oxyingenol and ingenol by using our synthetic strategy. In vitro activation assays of protein kinase C (PKC) α and δ revealed that the dodecanoyl group at O13 on 13-oxyingenol analogs had a significant role in PKCδ activation. The PKCα- or PKCδ-activating 13-oxyingenol and ingenol analogs induced both distinct morphological changes and increases of CD11b expression in HL-60 cells, which would be typical signs of HL-60 cell differentiation to macrophage-like cells, as expected by previous reports. Intriguingly, however, similar differentiation phenotypes were observed with the use of 13-oxyingenol natural derivatives and 13-oxyingenol-13-dodecanoate showing a remarkably less potent PKCα or PKCδ activation ability, which the PKC inhibitor Gö6983 diminished. This indicated the involvement of other PKC isozymes or related kinase activities. 13-Oxyingenol analogs, which induced HL-60 cell differentiation, also induced HL-60 cell death, similar to the action of a phorbol ester, a strong PKC activator.

Second-generation total synthesis of aplyronine A featuring Ni/Cr-mediated coupling reactions.

Second-generation total synthesis of aplyronine A, a potent antitumor marine macrolide, was achieved using Ni/Cr-mediated coupling reactions as key steps. The overall yield of the second-generation synthetic pathway of aplyronine A was 1.4%, obtained in 38 steps based on the longest linear sequence. Compared to our first-generation synthetic pathway of aplyronine A, the second-generation synthesis greatly improved both the yield and number of steps. In particular, we improved the stereoselectivity in the construction of the C13 stereogenic center and the C14-C15 (E)-trisubstituted double bond using the asymmetric Ni/Cr-mediated coupling reaction. Furthermore, we established efficient reaction conditions for the asymmetric Ni/Cr-mediated coupling reaction between the C21-C28 segment and C29-C34 segment. Thus, this coupling reaction proceeded with an equimolar ratio of each segment.

Analysis of the aplyronine A-induced protein-protein interaction between actin and tubulin by surface plasmon resonance.

The antitumor macrolide aplyronine A induces protein-protein interaction (PPI) between actin and tubulin to exert highly potent biological activities. The interactions and binding kinetics of these molecules were analyzed by the surface plasmon resonance with biotinylated aplyronines or tubulin as ligands. Strong binding was observed for tubulin and actin with immobilized aplyronine A. These PPIs were almost completely inhibited by one equivalent of either aplyronine A or C, or mycalolide B. In contrast, a non-competitive actin-depolymerizing agent, latrunculin A, highly accelerated their association. Significant binding was also observed for immobilized tubulin with an actin-aplyronine A complex, and the dissociation constant KD was 1.84µM. Our method could be used for the quantitative analysis of the PPIs between two polymerizing proteins stabilized with small agents.

Discovery of O6-benzyl glaziovianin A, a potent cytotoxic substance and a potent inhibitor of α,ß-tubulin polymerization.

We have discovered O6-benzyl glaziovianin A, which showed stronger inhibition of microtubule polymerization (IC50=2.1µM) than known α,ß-tubulin inhibitors, such as colchicine and glaziovianin A. Also, we performed competition binding experiments of O6-benzyl glaziovianin A and revealed that O6-benzyl glaziovianin A binds to the colchicine binding site with high affinity. It is interesting that glaziovianin A derivatives change their mode of action in benzylation at the O6 (α,ß-tubulin inhibitor) or O7 (γ-tubulin-specific inhibitor) position.

Marine natural products that interfere with multiple cytoskeletal protein interactions.

Various marine natural products that target cytoskeletal proteins have been discovered. A few of these compounds have recently been shown to induce or inhibit protein-protein interactions. Lobophorolide, an actin filament-disrupting macrolide, binds to actin with a unique 2 : 2 stoichiometry in which two lobophorolide molecules cooperate to stabilize an actin dimer. Adociasulfates, merotriterpenoid derivatives, inhibit microtubule-stimulated ATPase activity of a motor protein kinesin by blocking both the binding of microtubules and the processive motion of kinesin along microtubules. The antitumor macrolide aplyronine A synergistically binds to tubulin in association with actin, and prevents spindle formation and mitosis. In this highlight, we address recent chemical biology studies on these mechanistically-attractive marine natural products. These findings may be useful for the design and development of new pharmacological tools and therapeutic agents.

Synthesis and structure-activity relationships for cytotoxicity and apoptosis-inducing activity of (+)-halichonine B.

Halichonine B is a sesquiterpene alkaloid isolated from the marine sponge Halichondria okadai Kadota. Halichonine B has exhibited cytotoxicity against mammalian cancer cells and induced apoptosis in the human leukemia cell line HL60. Here we established a practical route for the synthesis of halichonine B and its analogues, and we evaluated their biological activities. It was revealed that the secondary amino groups in the side chain portion are important for the strong cytotoxicity of halichonine B and that the N(11)-prenyl group is unimportant. Halichonine B and its analogues were also observed to induce apoptosis in HL60 cells.

Aplysiasecosterol†A: A 9,11-Secosteroid with an Unprecedented Tricyclic γ-Diketone Structure from the Sea Hare Aplysia kurodai.

A new 9,11-secosteroid having an unprecedented tricyclic γ-diketone structure, aplysiasecosterol A (1), was isolated from the sea hare Aplysia kurodai. The structure was determined by one- and two-dimensional NMR spectroscopic analysis, molecular modeling studies, a comparison of experimental and calculated ECD spectra, and a modified Mosher's method. Aplysiasecosterol A (1) exhibited cytotoxicity against human myelocytic leukemia HL-60 cells. A biosynthetic pathway for 1 from a known cholesterol was proposed and includes twice α-ketol rearrangements and an intramolecular acetalization.

Total Synthesis of Mycalolides A and B through Olefin Metathesis.

An asymmetric total synthesis of the trisoxazole marine macrolides mycalolides A and B is described. This synthesis involves the convergent assembly of highly functionalized C1-C19 trisoxazole and C20-C35 side-chain segments through the use of olefin metathesis and esterification as well as Julia-Kocienski olefination and enamide formation as key steps.

Discovery of 1-oxa-4,9-diazaspiro[5.5]undecane-based trisubstituted urea derivatives as highly potent soluble epoxide hydrolase inhibitors and orally active drug candidates for treating of chronic kidney diseases.

We identified 1-oxa-4,9-diazaspiro[5.5]undecane-based trisubstituted ureas as highly potent soluble epoxide hydrolase (sEH) inhibitors and orally active agents for treating chronic kidney diseases. Compound 19 exhibited excellent sEH inhibitory activity and bioavailability. When administered orally at 30 mg/kg, 19 lowered serum creatinine in a rat model of anti-glomerular basement membrane glomerulonephritis but 2,8-diazaspiro[4.5]decane-based trisubstituted ureas did not. These results suggest that 19 is an orally active drug candidate for treating chronic kidney diseases.

Concise total synthesis of (-)-berkelic acid via regioselective spiroacetal/pyran formation.

We successfully developed (1) scalable synthesis of the triol segment and (2) regio- and stereoselective synthesis of the tetracyclic skeleton by tandem spiroacetal/pyran formation from a simpler alkyne precursor, resulting in the achievement of concise total synthesis of (-)-berkelic acid.

Inhibition of microtubule assembly by a complex of actin and antitumor macrolide aplyronine A.

Aplyronine A (ApA) is a marine natural product that shows potent antitumor activity. While both ApA and ApC, a derivative of ApA that lacks a trimethylserine ester moiety, inhibit actin polymerization in vitro to the same extent, only ApA shows potent cytotoxicity. Therefore, the molecular targets and mechanisms of action of ApA in cells have remained unclear. We report that ApA inhibits tubulin polymerization in a hitherto unprecedented way. ApA forms a 1:1:1 heterotrimeric complex with actin and tubulin, in association with actin synergistically binding to tubulin, and inhibits tubulin polymerization. Tubulin-targeting agents have been widely used in cancer chemotherapy, but there are no previous descriptions of microtubule inhibitors that also bind to actin and affect microtubule assembly. ApA inhibits spindle formation and mitosis in HeLa S3 cells at 100 pM, a much lower concentration than is needed for the disassembly of the actin cytoskeleton. The results of the present study indicate that ApA represents a rare type of natural product, which binds to two different cytoplasmic proteins to exert highly potent biological activities.

Apoptosis-inducing activity of the actin-depolymerizing agent aplyronine A and its side-chain derivatives.

Aplyronine A (1) and mycalolide B (2), which are cytotoxic actin-depolymerizing marine macrolides, were revealed to induce apoptosis in human leukemia HL60 cells and human epithelial carcinoma HeLa S(3) cells. Based on these results, actin-depolymerizing compounds were expected to exhibit apoptosis-inducing activity in cancer cells. Compounds 3-6, which were synthesized based on the side-chain structure of aplyronine A, were evaluated for their actin-depolymerizing activities in vitro and cytotoxicities against HL60 cells. The growth-inhibitory activities of 3-6 were well correlated with their actin-depolymerizing activities, and derivative 6 was shown to induce the disruption of actin filaments and apoptosis in HL60 cells. These results suggested that actin-depolymerizing agents 1, 2, and 6-induced apoptosis in HL60 cells may have been due to their actin-depolymerizing activity.